EP3054005B1 - Heparosanproduzierendes bakterium und heparosanherstellungsverfahren - Google Patents

Heparosanproduzierendes bakterium und heparosanherstellungsverfahren Download PDF

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EP3054005B1
EP3054005B1 EP14850420.2A EP14850420A EP3054005B1 EP 3054005 B1 EP3054005 B1 EP 3054005B1 EP 14850420 A EP14850420 A EP 14850420A EP 3054005 B1 EP3054005 B1 EP 3054005B1
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escherichia coli
strain
gene
heparosan
protein
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EP3054005A4 (de
EP3054005A1 (de
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Shunsuke Yamazaki
Tomoko Shimizu
Kenichi Mori
Naoto Tonouchi
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Ajinomoto Co Inc
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Ajinomoto Co Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • the present invention relates to a heparosan-producing bacterium and a method for producing heparosan.
  • Heparosan (also referred to as N-acetylheparosan) is a polysaccharide constituted by a repetition structure of a disaccharide consisting of a glucuronic acid (GlcUA) residue and an N-acetyl-D-glucosamine (GlcNAc) residue [ ⁇ 4)- ⁇ -GlcUA-(1 ⁇ 4)- ⁇ -GlcNAc-(1 ⁇ ].
  • heparosan is produced by the Escherichia coli K5 strain and the Pasteurella multocida type D strain as a capsular polysaccharide (Non-patent document 1). These heparosan-producing bacteria show pathogenicity causing urinary tract infections, atrophic rhinitis, etc. for mammals.
  • glucosyltransferases which are heparosan synthetases, and six kinds of heparosan efflux carriers are required for the biosynthesis of heparosan. That is, GlcNAc and GlcUA are first alternately added to a non-reducing end of sugar chain by the glucosyltransferases (KfiA and KfiC), and the heparosan chain is thereby extended (Non-patent document 2).
  • Non-patent document 3 the heparosan efflux carriers
  • KpsC, KpsD, KpsE, KpsM, KpsS, and KpsT the heparosan efflux carriers
  • the heparosan chain is fixed to a phosphatidic acid molecule in the outer membrane of Escherichia coli on the cell surface through lipid substitution at the reducing end (Non-patent document 4).
  • the heparosan synthetase genes and the heparosan efflux carrier genes form a cluster on the chromosome.
  • the cluster is divided into the regions 1 to 3, and the region 2 locating at the center of the cluster encodes the four proteins including the heparosan synthetases (KfiA, KfiB, KfiC, and KfiD).
  • the Pasteurella multocida type D strain has PmHS1 as a heparosan synthetase (glucosyltransferase) (Non-patent document 5).
  • PmHS1 has active domains homologous to both KfiA and KfiC of the Escherichia coli K5 strain, and it catalyzes a polymerization reaction using both UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates.
  • any heparosan efflux carrier of the Pasteurella multocida type D strain has not been clarified yet.
  • Heparin is one of anticoagulants, and is used for therapeutic treatments of thromboembolism and disseminated intravascular coagulation (DIC), prevention of blood coagulation at the time of artificial dialysis and extracorporeal circulation, and so forth.
  • Heparosan is a sugar chain structure of heparin, and can be converted into a heparin-like polysaccharide through such steps as deacetylation, epimerization, sulfation, and molecular weight adjustment (Non-patent documents 6 and 7).
  • Heparin exhibits an anticoagulant activity through activation of antithrombin III, which is an anticoagulant.
  • Antithrombin III binds to the active serine moieties of thrombin, Xa factor (active type of X factor), and other serine proteases to inhibit them.
  • Thrombin is a blood coagulation factor
  • the Xa factor is a factor involved in the maturation of thrombin. Heparin binds to this antithrombin III to change the structure thereof, and thereby activates the inhibitory activity.
  • Thrombin shows higher affinity for the heparin-antithrombin-III complex compared with the Xa factor.
  • Non-patent documents 6 and 7 heparosan obtained from the Escherichia coli K5 strain can be enzymatically converted into a heparin-like anticoagulant polysaccharide. Further, heparosan can also be utilized in various uses besides the manufacture of heparin (Patent document 1).
  • Non-patent document 9 a heparosan-producing bacterium produced from a nonpathogenic Escherichia coli BL21(DE3) strain as a host strain has very recently been reported (Non-patent document 9). That is, in flask culture of the BL21 strain introduced with an expression vector pETDuet-1 carrying the four heparosan biosynthesis genes, kfiA, kfiB, kfiC, and kfiD, which constitute the region 2 of the Escherichia coli K5 strain, production of 334 mg/L of heparosan was confirmed.
  • Objects of the present invention are to develop a novel technique for improving heparosan-producing ability of bacteria, and thereby provide an efficient method for producing heparosan.
  • the inventors of the present invention performed various researches, as a result, found that, by increasing expression of one or more kinds of genes selected from the genes mentioned in Tables 1 to 3 in bacteria having a heparosan-producing ability, the heparosan-producing ability could be improved.
  • the present invention relates to:
  • RpoE is sigma E ( ⁇ E ), which is one of the sigma factors and functions as a subunit of RNA polymerase. RpoE controls expression of protease against membrane and intermembrane proteins in response to heat shock and stress ( Ades S.E. et al. (2003) "Regulation of the alternative sigma factor sigma(E) during initiation, adaptation, and shutoff of the extracytoplasmic heat shock response in Escherichia coli", J. Bacteriol., 185(8):2512-9 ). There is no finding at all indicating relevance of this protein and heparosan production.
  • the bacterium of the present invention is an Escherichia bacterium having a heparosan-producing ability that has been modified so that expression of the rpoE gene is increased as compared with that of a non-modified strain.
  • the "bacterium having a heparosan-producing ability” refers to a bacterium having an ability to produce and accumulate heparosan in a medium in such a degree that heparosan can be collected, when the bacterium is cultured in the medium.
  • the bacterium having a heparosan-producing ability may be a bacterium that is able to accumulate heparosan in a medium in an amount larger than that obtainable with a non-modified strain. Examples of the non-modified strain include wild strains and the parent strain of the bacterium.
  • the bacterium having a heparosan-producing ability may be a bacterium that is able to accumulate heparosan in a medium in an amount of, for example, 50 mg/L or more, 100 mg/L or more, 200 mg/L or more, or 300 mg/L or more.
  • the Escherichia bacterium is not particularly limited, and examples thereof include those classified into the genus Escherichia according to the taxonomy known to those skilled in the field of microbiology.
  • Examples of the Escherichia bacterium include, for example, those described in the work of Neidhardt et al. ( Backmann B.J., 1996, Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, pp.2460-2488 , Table 1, In F.D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology, Second Edition, American Society for Microbiology Press, Washington, D.C .).
  • Escherichia bacterium examples include, for example, Escherichia coli.
  • Escherichia coli include, for example, Escherichia coli K-12 strains such as Escherichia coli W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); Escherichia coli B strains such as BL21(DE3) strain; and derivative strains thereof.
  • strains are available from, for example, the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852, P.O. Box 1549, Manassas, VA 20108, United States of America). That is, registration numbers are given to the respective strains, and the strains can be ordered by using these registration numbers (refer to http://www.atcc.org/). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.
  • the BL21(DE3) strain is also available from, for example, Life Technologies (product number C6000-03).
  • the bacterium of the present invention may be a bacterium inherently having a heparosan-producing ability, or may be a bacterium modified so that it has a heparosan-producing ability.
  • the bacterium having a heparosan-producing ability can be obtained by, for example, imparting a heparosan-producing ability to such a bacterium as mentioned above.
  • a heparosan-producing ability can be imparted by introducing a gene encoding a protein that participates in heparosan production.
  • a protein that participates in heparosan production include glycosyltransferase and heparosan efflux carrier protein.
  • one kind of gene or two or more kinds of genes may be introduced.
  • a gene may be introduced in the same manner as that of the method of increasing copy number of gene described later.
  • glycosyltransferase referred to herein means a protein having an activity for catalyzing a reaction of adding N-acetyl-D-glucosamine (GlcNAc) and/or glucuronic acid (GlcUA) to a non-reducing end of a sugar chain to thereby extend a heparosan chain. This activity is also referred to as “glycosyltransferase activity”.
  • Examples of the gene encoding glycosyltransferase include the kfiA gene, kfiC gene, and pmHS1 gene.
  • Examples of the kfiA gene and kfiC gene include the kfiA gene and kfiC gene of the Escherichia coli K5 strain.
  • the KfiA protein encoded by the kfiA gene of the Escherichia coli K5 strain adds GlcNAc to a non-reducing end of a sugar chain by using UDP-GlcNAc as a substrate.
  • the KfiC protein encoded by the kfiC gene of the Escherichia coli K5 strain adds GlcUA to a non-reducing end of a sugar chain by using UDP-GlcUA as a substrate.
  • the kfiA and kfiC genes of the Escherichia coli K5 strain constitute the kfiABCD operon (also referred to as region 2) together with the kfiB and kfiD genes.
  • the nucleotide sequence of a region containing the kfiABCD operon of the Escherichia coli K5 strain is shown as SEQ ID NO: 24.
  • the kfiA, kfiB, kfiC, and kfiD genes correspond to the sequence of the positions 445 to 1,164, the sequence of the positions 1,593 to 3,284, the sequence of the positions 4,576 to 6,138, and the sequence of the positions 6,180 to 7,358, respectively.
  • the amino acid sequences of KfiA, KfiB, KfiC, and KfiD proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 25 to 28, respectively.
  • Examples of the pmHS1 gene include the pmHS1 gene of the Pasteurella multocida type D strain.
  • the PmHS1 protein encoded by the pmHS1 gene of the Pasteurella multocida type D strain alternately adds GlcNAc and GlcUA to a non-reducing end of a sugar chain by using both UDP-GlcNAc and UDP-GlcUA as substrates.
  • the nucleotide sequence of the pmHS1 gene of the Pasteurella multocida type D strain and the amino acid sequence of the protein encoded by this gene can be obtained from public databases such as the NCBI database (http://www.ncbi.nlm.nih.gov/).
  • heparosan efflux carrier protein means a protein having the activity of excreting a heparosan chain out of a cell through cell membranes. This activity is also referred to as “heparosan efflux activity”. Examples of gene encoding the heparosan efflux carrier protein include the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes.
  • Examples of the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes include the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes of the Escherichia coli K5 strain and Escherichia coli B strain.
  • the kpsC, kpsD, kpsE, and kpsS genes of these strains constitute the kpsFEDUCS operon (also referred to as region 1) together with the kpsF and kpsU genes.
  • the kpsM and kpsT genes of these strains constitute the kpsMT operon (also referred to as region 3).
  • the nucleotide sequences of the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes of these strains, and the amino acid sequences of the proteins encoded by these genes can be obtained from public databases such as the NCBI database (http://www.ncbi.nlm.nih.gov/).
  • the gene to be introduced can be appropriately chosen according to type of the bacterium to be used, and so forth.
  • the Escherichia coli B strain has genes encoding a heparosan efflux carrier protein, but it does not have genes encoding glycosyltransferase. Therefore, a heparosan-producing ability can be imparted to the Escherichia coli B strain by introducing gene(s) encoding glycosyltransferase.
  • the Escherichia coli K-12 strain does not have either genes encoding glycosyltransferase or genes encoding a heparosan efflux carrier protein.
  • a heparosan-producing ability can be imparted to the Escherichia coli K-12 strain by introducing both gene(s) encoding glycosyltransferase and genes encoding a heparosan efflux carrier protein.
  • examples of the Escherichia bacterium having a heparosan-producing ability include, for example, Escherichia coli K5 strain; Escherichia coli B strain such as BL21(DE3) strain introduced with the kfiA gene and kfiC gene of the Escherichia coli K5 strain; Escherichia coli K-12 strain such as W3110 strain and MG1655 strain introduced with the kfiA gene and kfiC gene of the Escherichia coli K5 strain, as well as the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes of the Escherichia coli K5 strain or Escherichia coli B strain; and derivative strains thereof.
  • Escherichia coli B strain introduced with the kfiA gene and kfiC gene of the Escherichia coli K5 strain include, for example, the Escherichia coli BL21(DE3)/pVK9-region2 described in Examples.
  • the bacterium having a heparosan-producing ability may also be a bacterium modified so that expression of a gene inherently possessed by the bacterium among genes encoding a protein involved in the heparosan production is enhanced. That is, for example, the Escherichia coli K5 strain may be modified so that expression of one or more kinds of genes encoding a protein that participates in the heparosan production is enhanced. Further, for example, the Escherichia coli B strain may be modified so that expression of one or more kinds of genes encoding a heparosan efflux carrier protein is enhanced.
  • the bacterium having a heparosan-producing ability may have been further modified in another way so long as the heparosan-producing ability is not degraded.
  • the bacterium having a heparosan-producing ability may have been modified so that expression of one or more kinds of genes selected from kfiB, kfiD, kpsF, and kpsU genes is enhanced. That is, when genes encoding glycosyltransferase are introduced, for example, the region 2 may be totally introduced, and when genes encoding glycosyltransferase and genes encoding a heparosan efflux carrier protein are introduced, the regions 1 to 3 may be totally introduced.
  • the gene used for modification of a bacterium such as impartation of a heparosan-producing ability is not limited to the genes exemplified above or genes having a known nucleotide sequence, but may be a variant of such genes, so long as the variant encodes a protein that maintains the original function.
  • the expression "protein maintains the original function” means that, in the case of the glycosyltransferase, for example, the variant of the protein has the glycosyltransferase activity, or in the case of the heparosan efflux carrier protein, the variant of the protein has the heparosan efflux activity.
  • the gene used for modification of the bacterium such as impartation of a heparosan-producing ability may be a gene encoding a protein having a known amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions.
  • the descriptions for conservative variants of the genes mentioned in Tables 1 to 3 and the proteins encoded by them can be mutatis mutandis applied.
  • the bacterium of the present invention has been modified so that expression of the rpoE gene is increased as compared with that of a non-modified strain.
  • the bacterium of the present invention can be obtained by modifying a bacterium having a heparosan-producing ability so that expression of the rpoE gene is increased as compared with that of a non-modified strain.
  • the bacterium of the present invention can also be obtained by modifying a bacterium so that expression of the rpoE gene is increased as compared with that of a non-modified strain, and then imparting a heparosan-producing ability to the bacterium.
  • the bacterium of the present invention may be a bacterium that has acquired a heparosan-producing ability as a result of the modification for increasing expression of the rpoE gene.
  • modifications for constructing the bacterium of the present invention can be performed in an arbitrary order.
  • the "genes mentioned in Tables 1 to 3" are, specifically, rbsR, rbsK, rbsB, hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB, rfaH, nusG, pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY, yjtD, thrL, thrA, thrB
  • the rpoE gene is a gene encoding SigmaE ( ⁇ E ).
  • the rpoE gene of the Escherichia coli K-12 MG1655 strain corresponds to the complementary sequence of the sequence of the positions 2,707,459 to 2,708,034 in the genome sequence registered at the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990).
  • GenBank accession NC_000913 VERSION NC_000913.2 GI: 49175990.
  • the RpoE protein of the MG1655 strain is registered as GenBank accession NP_417068 (version NP_417068.1 GI: 16130498).
  • the bacterium of the present invention may have been also modified so that, for example, expression of at least the rpoE gene among the genes of Tables 1 to 3 is increased.
  • the combination of the genes of Tables 1 to 3 of which expression is to be increased is not particularly limited. Examples of the combination include, for example, the combinations described in Examples mentioned later.
  • Expression of the gene(s) of Tables 1 to 3 may be increased by, for example, increasing the copy number of a DNA containing the gene(s) of Tables 1 to 3, such as a DNA having the nucleotide sequence shown as SEQ ID NO: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, or 162.
  • a DNA containing the gene(s) of Tables 1 to 3 such as a DNA having the nucleotide sequence shown as SEQ ID NO: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, or 162.
  • the copy number of a DNA containing a part of the irp gene may also be increased.
  • DNA as mentioned above of which the copy number is to be increased may be a variant of a DNA having the nucleotide sequence shown as SEQ ID NO: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, or 162.
  • the descriptions about conservative variants of the genes mentioned in Tables 1 to 3 can be mutatis mutandis applied. Namely, for example, the copy number of a DNA showing a homology of 90% or more to the nucleotide sequence shown as SEQ ID NOS: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, or 162 may be increased.
  • genes can be obtained by PCR using chromosome of a strain having any of these genes as the template, and oligonucleotides produced on the basis of any of these known gene sequences as the primers.
  • the genes of Tables 1 to 3 each may be a variant of the genes exemplified above, so long as the variant maintains the original function.
  • the proteins encoded by the genes of Tables 1 to 3 each may be a variant of the proteins exemplified above, so long as the variant maintains the original function.
  • Such a variant that maintains the original function may be referred to as "conservative variant”.
  • the genes specified with the aforementioned gene names and the proteins specified with names corresponding to the gene names include the genes and proteins exemplified above, respectively, and in addition, conservative variants thereof.
  • the term " rpoE gene” includes the rpoE genes exemplified above (i.e.
  • the term "RpoE protein” includes the RpoE proteins exemplified above (i.e. RpoE proteins of the Escherichia coli K-12 MG1655 strain and the Escherichia coli K5 strain), and in addition, conservative variants thereof.
  • the conservative variants include, for example, homologues and artificially modified variants of the genes and proteins exemplified above.
  • variant maintains the original function means that the variant of a gene or protein has a function (such as activity or property) corresponding to the function (such as activity or property) of the original gene or protein.
  • the expression “variant maintains the original function” means that, in the case of the genes of Tables 1 to 3, a variant of any of the genes has a property of increasing heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount of the variant is increased in the bacterium. Further, the expression “variant maintains the original function” may also mean that, in the case of the genes of Tables 1 to 3, a variant of any of the genes encodes a protein that maintains the original function. That is, the genes of Tables 1 to 3 may encode a conservative variant of the proteins exemplified above.
  • the expression “variant maintains the original function” means that, in the case of the proteins encoded by the genes of Tables 1 to 3, a variant of any of the proteins has a property of increasing heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount of the variant is increased in the bacterium. Further, the expression “variant maintains the original function” may also mean that, in the case of the proteins encoded by the genes of Tables 1 to 3, a variant of any of the proteins has the above-mentioned function of the corresponding protein, for example, the function of the sigmaE ( ⁇ E ) in the case of the RpoE protein.
  • Whether a variant of a gene or protein has the property of increasing heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium can be confirmed by introducing the gene or a gene encoding the protein into the Escherichia bacterium having heparosan-producing ability, and confirming whether the heparosan-producing ability is improved or not.
  • homologues of the genes of Tables 1 to 3 can be easily obtained from public databases by, for example, BLAST search or FASTA search using any of the nucleotide sequences of the genes exemplified above as a query sequence. Further, homologues of the genes of Tables 1 to 3 can also be obtained by, for example, PCR using a chromosome of a microorganism such as bacterium as the template, and oligonucleotides prepared on the basis of any of these known gene sequences as the primers.
  • the genes of Tables 1 to 3 each may encode a protein having any of the aforementioned amino acid sequences including substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, so long as the protein maintains the original function.
  • the N-terminus and/or C-terminus of the encoded protein may be extended or shortened.
  • the number of "one or several" may differ depending on the positions in the three-dimensional structure of the protein or the types of amino acid residues, specifically, it is, for example, 1 to 50, 1 to 40, or 1 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.
  • the aforementioned substitution, deletion, insertion, or addition of one or several amino acid residues is a conservative mutation that maintains normal function of the protein.
  • Typical examples of the conservative mutation are conservative substitutions.
  • the conservative substitution is a mutation wherein substitution takes place mutually among Phe, Trp, and Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile, and Val, if it is a hydrophobic amino acid; between Gln and Asn, if it is a polar amino acid; among Lys, Arg, and His, if it is a basic amino acid; between Asp and Glu, if it is an acidic amino acid; and between Ser and Thr, if it is an amino acid having a hydroxyl group.
  • substitutions considered as conservative substitutions include, specifically, substitution of Ser or Thr for Ala, substitution of Gln, His, or Lys for Arg, substitution of Glu, Gln, Lys, His, or Asp for Asn, substitution of Asn, Glu, or Gln for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp, or Arg for Gln, substitution of Gly, Asn, Gln, Lys, or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg, or Tyr for His, substitution of Leu, Met, Val, or Phe for Ile, substitution of Ile, Met, Val, or Phe for Leu, substitution of Asn, Glu, Gln, His, or Arg for Lys, substitution of Ile, Leu, Val, or Phe for Met, substitution of Trp, Tyr, Met, Ile, or Leu for Phe, substitution of Thr or Ala for Ser
  • genes of Tables 1 to 3 each may be a gene encoding a protein showing a homology of, for example, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, to the total amino acid sequence of any of the amino acid sequences mentioned above, so long as the protein maintains the original function.
  • "homology” may mean "identity”.
  • the genes of Tables 1 to 3 each may also be a DNA that is able to hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to a partial or entire sequence of any of the aforementioned nucleotide sequences.
  • stringent conditions refer to conditions under which a so-called specific hybrid is formed, and a non-specific hybrid is not formed.
  • Examples of the stringent conditions include those under which highly homologous DNAs hybridize to each other, for example, DNAs not less than 80% homologous, preferably not less than 90% homologous, more preferably not less than 95% homologous, still more preferably not less than 97% homologous, particularly preferably not less than 99% homologous, hybridize to each other, and DNAs less homologous than the above do not hybridize to each other, or conditions of washing of typical Southern hybridization, i.e., conditions of washing once, preferably 2 or 3 times, at a salt concentration and temperature corresponding to 1 x SSC, 0.1% SDS at 60°C, preferably 0.1 x SSC, 0.1% SDS at 60°C, more preferably 0.1 x SSC, 0.1% SDS at 68°C.
  • the probe used for the aforementioned hybridization may be a part of a sequence that is complementary to a gene.
  • a probe can be prepared by PCR using oligonucleotides prepared on the basis of a known gene sequence as the primers and a DNA fragment containing any of the genes of Tables 1 to 3 as the template.
  • a DNA fragment having a length of about 300 bp can be used as the probe.
  • the washing conditions of the hybridization may be, for example, 50°C, 2 x SSC and 0.1% SDS.
  • the genes of Tables 1 to 3 each may be a gene in which an arbitrary codon is replaced with an equivalent codon, so long as the original function is maintained.
  • the genes of Tables 1 to 3 each may be modified so that they have optimal codons according to codon usage of the host.
  • a variant of the genes of Tables 1 to 3 can be obtained by, for example, modifying a coding region of the genes by site-specific mutagenesis so that a specific site of the encoded protein include substitution, deletion, insertion, or addition of amino acid residues. Further, a variant of the genes of Tables 1 to 3 can also be obtained by the conventionally known mutagenesis.
  • Examples of the mutagenesis include such methods as treating a DNA molecule having a nucleotide sequence of any of the genes of Tables 1 to 3 in vitro with hydroxylamine or the like, irradiating X-ray or ultraviolet ray on a microorganism such as a microorganism belonging to Enterobacteriaceae containing any of the genes of Tables 1 to 3, treating such a microorganism with a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS), performing error prone PCR ( Cadwell, R.C., PCR Meth.
  • NTG N-methyl-N'-nitro-N-nitrosoguanidine
  • EMS ethyl methanesulfonate
  • MMS methyl methanesulfonate
  • the expression of a gene may be increased 1.5 times or more, 2 times or more, or 3 times or more, as compared with that of a non-modified strain.
  • the state that "the expression of a gene is increased” includes not only a state that the expression amount of an objective gene is increased in a strain that inherently expresses the objective gene, but also a state that the gene is introduced into a strain that does not inherently express the objective gene, and expressed therein. That is, the phrase "the expression of a gene is increased” may also mean, for example, that an objective gene is introduced into a strain that does not possess the gene, and is expressed therein.
  • the state that "the expression of a gene is increased” may also be referred to as "the expression of a gene is enhanced”.
  • the expression of a gene can be increased by, for example, increasing the copy number of the gene.
  • the copy number of a gene can be increased by introducing the gene into the chromosome of a host.
  • a gene can be introduced into a chromosome by, for example, using homologous recombination ( Miller, J.H., Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory ). Only one copy, or two or more copies of a gene may be introduced.
  • homologous recombination Miller, J.H., Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory
  • Only one copy, or two or more copies of a gene may be introduced.
  • by performing homologous recombination using a sequence which is present in multiple copies on a chromosome as a target multiple copies of a gene can be introduced into the chromosome. Examples of such a sequence which is present in multiple copies on a chromosome include repetitive DNAs, and inverted repeats located at the both ends of a transposon.
  • homologous recombination may be performed by using an appropriate sequence on a chromosome such as a gene unnecessary for the production of an objective substance as a target.
  • Homologous recombination can be performed by, for example, a method using a linear DNA such as Red-driven integration ( Datsenko, K.A., and Wanner, B.L., Proc. Natl. Acad. Sci.
  • a gene can also be randomly introduced into a chromosome by using a transposon or Mini-Mu (Japanese Patent Laid-open (Kokai) No. 2-109985 , U.S. Patent No. 5,882,888 , EP 805867 B1 ).
  • a target gene into a chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to the whole gene or a part thereof, PCR using primers prepared on the basis of the sequence of the gene, or the like.
  • the copy number of a gene can also be increased by introducing a vector containing the gene into a host.
  • the copy number of a target gene can be increased by ligating a DNA fragment containing the target gene with a vector that functions in a host to construct an expression vector of the gene, and transforming the host with the expression vector.
  • the DNA fragment containing the target gene can be obtained by, for example, PCR using the genomic DNA of a microorganism having the target gene as the template.
  • a vector autonomously replicable in the cell of the host can be used as the vector.
  • the vector is preferably a multi-copy vector. Further, the vector preferably has a marker such as an antibiotic resistance gene for selection of transformant.
  • the vector may have a promoter and/or terminator for expressing the introduced gene.
  • the vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, cosmid, phagemid, or the like.
  • vector autonomously replicable in Enterobacteriaceae bacteria such as Escherichia coli
  • Escherichia coli include, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all of these are available from Takara Bio), pACYC184, pMW219 (NIPPON GENE), pTrc99A (Pharmacia), pPROK series vectors (Clontech), pKK233-2 (Clontech), pET series vectors (Novagen), pQE series vectors (QIAGEN), and the broad host spectrum vector RSF1010.
  • the gene When a gene is introduced, it is sufficient that the gene is expressibly harbored by the bacterium of the present invention. Specifically, it is sufficient that the gene is introduced so that it is expressed under control by a promoter sequence that functions in the bacterium of the present invention.
  • the promoter may be a promoter derived from the host, or a heterogenous promoter.
  • the promoter may be the native promoter of the gene to be introduced, or a promoter of another gene. As the promoter, for example, such a stronger promoter as mentioned later may also be used.
  • a terminator for termination of gene transcription may be located downstream of the gene.
  • the terminator is not particularly limited so long as it functions in the bacterium of the present invention.
  • the terminator may be a terminator derived from the host, or a heterogenous terminator.
  • the terminator may be the native terminator of the gene to be introduced, or a terminator of another gene. Specific examples of the terminator include, for example, T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.
  • genes each are expressibly harbored by the bacterium of the present invention.
  • all the genes may be carried by a single expression vector or a chromosome.
  • the genes may be separately carried by two or more expression vectors, or separately carried by a single or two or more expression vectors and a chromosome.
  • An operon constituted by two or more genes may also be introduced.
  • the case of "introducing two or more genes” include, for example, introducing respective genes coding for two or more kinds of enzymes, introducing respective genes coding for two or more subunits constituting a single enzyme, and a combination of the foregoing cases.
  • the gene to be introduced is not particularly limited so long as it codes for a protein that functions in the host.
  • the gene to be introduced may be a gene derived from the host, or may be a heterogenous gene.
  • the gene to be introduced can be obtained by, for example, PCR using primers designed on the basis of the nucleotide sequence of the gene, and using the genomic DNA of an organism having the gene, a plasmid carrying the gene, or the like as a template.
  • the gene to be introduced may also be totally synthesized, for example, on the basis of the nucleotide sequence of the gene ( Gene, 60(1), 115-127 (1987 )).
  • a part or all of the plurality of subunits may be modified, so long as the activity of the protein is eventually increased. That is, for example, when the activity of a protein is increased by increasing the expression of a gene, the expression of a part or all of the plurality of genes that code for the subunits may be enhanced. It is usually preferable to enhance the expression of all of the plurality of genes coding for the subunits.
  • the subunits constituting the complex may be derived from a single kind of organism or two or more kinds of organisms, so long as the complex has a function of the objective protein. That is, for example, genes of the same organism coding for a plurality of subunits may be introduced into a host, or genes of different organisms coding for a plurality of subunits may be introduced into a host.
  • the expression of a gene can be increased by improving the transcription efficiency of the gene.
  • the transcription efficiency of a gene can be improved by, for example, replacing the promoter of the gene on a chromosome with a stronger promoter.
  • the "stronger promoter" means a promoter providing an improved transcription of a gene compared with an inherently existing wild-type promoter of the gene. Examples of stronger promoters include, for example, the known high expression promoters such as T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter.
  • a highly-active type of an existing promoter may also be obtained by using various reporter genes. For example, by making the -35 and -10 regions in a promoter region closer to the consensus sequence, the activity of the promoter can be enhanced ( WO00/18935 ).
  • highly active-type promoter include various tac-like promoters (Katashkina JI et al., Russian Federation Patent Application No. 2006134574 ) and pnlp8 promoter ( WO2010/027045 ). Methods for evaluating the strength of promoters and examples of strong promoters are described in the paper of Goldstein et al. (Prokaryotic Promoters in Biotechnology, Biotechnol. Annu. Rev., 1, 105-128 (1995 )), and so forth.
  • the expression of a gene can also be increased by improving the translation efficiency of the gene.
  • the translation efficiency of a gene can be improved by, for example, replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) for the gene on a chromosome with a stronger SD sequence.
  • SD Shine-Dalgarno
  • RBS ribosome binding site
  • the "stronger SD sequence” means a SD sequence that provides an improved translation of mRNA compared with the inherently existing wild-type SD sequence of the gene. Examples of stronger SD sequences include, for example, RBS of the gene 10 derived from phage T7 ( Olins P.O. et al, Gene, 1988, 73, 227-235 ).
  • expression control region sites that affect the expression of a gene, such as promoter, SD sequence, and spacer region between RBS and the start codon, may also be collectively referred to as "expression control region".
  • Expression control regions can be identified by using a promoter search vector or gene analysis software such as GENETYX. These expression control regions can be modified by, for example, a method of using a temperature sensitive vector, or the Red driven integration method ( WO2005/010175 ).
  • the translation efficiency of a gene can also be improved by, for example, modifying codons.
  • Escherichia coli etc. a clear codon bias exists among the 61 amino acid codons found within the population of mRNA molecules, and the level of cognate tRNA appears directly proportional to the frequency of codon usage ( Kane, J.F., Curr. Opin. Biotechnol., 6 (5), 494-500 (1995 )). That is, if there is a large amount of mRNA containing an excess amount of rare codons, a translational problem may arise.
  • clusters of AGG/AGA, CUA, AUA, CGA, or CCC codons may especially reduce both the quantity and quality of a synthesized protein.
  • Such a problem occurs especially at the time of expression of a heterologous gene. Therefore, in the case of heterogenous expression of a gene or the like, the translation efficiency of the gene can be improved by replacing a rare codon present in the gene with a synonymous codon more frequently used. Codons can be replaced by, for example, the site-specific mutation method for introducing an objective mutation into an objective site of DNA. Examples of the site-specific mutation method include the method utilizing PCR ( Higuchi, R., 61, in PCR Technology, Erlich, H.A.
  • the expression of a gene can also be increased by amplifying a regulator that increases the expression of the gene, or deleting or attenuating a regulator that reduces the expression of the gene.
  • Such methods for increasing the gene expression as mentioned above may be used independently or in an arbitrary combination.
  • the method for the transformation is not particularly limited, and conventionally known methods can be used. There can be used, for example, a method of treating recipient cells with calcium chloride so as to increase the permeability thereof for DNA, which has been reported for the Escherichia coli K-12 strain ( Mandel, M. and Higa, A., J. Mol. Biol., 1970, 53, 159-162 ), and a method of preparing competent cells from cells which are in the growth phase, followed by transformation with DNA, which has been reported for Bacillus subtilis ( Duncan, C.H., Wilson, G.A. and Young, F.E., Gene, 1977, 1:153-167 ).
  • DNA-recipient cells into protoplasts or spheroplasts, which can easily take up recombinant DNA, followed by introducing a recombinant DNA into the DNA-recipient cells, which is known to be applicable to Bacillus subtilis, actinomycetes, and yeasts ( Chang, S. and Choen, S.N., 1979, Mol. Gen. Genet., 168:111-115 ; Bibb, M.J., Ward, J.M. and Hopwood, O.A., 1978, Nature, 274:398-400 ; Hinnen, A., Hicks, J.B. and Fink, G.R., 1978, Proc. Natl. Acad. Sci. USA, 75:1929-1933 ).
  • the electric pulse method reported for coryneform bacteria Japanese Patent Laid-open (Kokai) No. 2-207791
  • Japanese Patent Laid-open (Kokai) No. 2-207791 Japanese Patent Laid-open (K
  • An increase in the expression of a gene can be confirmed by confirming an increase in the transcription amount of the gene, or by confirming an increase in the amount of a protein expressed from the gene.
  • An increase in the expression of a gene can also be confirmed by confirming an increase in the activity of a protein expressed from the gene.
  • An increase of the transcription amount of a gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a non-modified strain such as a wild-type strain or parent strain.
  • a non-modified strain such as a wild-type strain or parent strain.
  • Examples of the method for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and so forth ( Sambrook, J., et al., Molecular Cloning A Laboratory Manual/Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001 ).
  • the amount of mRNA may increase, for example, 1.5 times or more, 2 times or more, or 3 times or more, as compared with that of a non-modified strain.
  • the amount of a protein can be confirmed by Western blotting using antibodies ( Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001 ).
  • the amount of the protein may increase, for example, 1.5 times or more, 2 times or more, or 3 times or more, as compared with that of a non-modified strain.
  • An increase in the activity of a protein can be confirmed by measuring the activity of the protein.
  • the activity of the protein may increase, for example, 1.5 times or more, 2 times or more, or 3 times or more, as compared with that of a non-modified strain.
  • the aforementioned methods for increasing the expression of a gene can be used for enhancement of the expression of arbitrary genes such as the genes of Tables 1 to 3 and genes encoding a protein that participates in heparosan production.
  • the method for producing heparosan of the present invention is a method for producing heparosan comprising culturing the bacterium of the present invention in a medium to produce and accumulate heparosan in the medium, and collecting heparosan from the medium.
  • the medium to be used is not particularly limited, so long as the bacterium of the present invention can proliferate in the medium, and heparosan is produced and accumulated.
  • a usual medium used for culture of bacteria can be used.
  • Specific examples of the medium include, for example, the LB medium (Luria-Bertani medium, containing 10.0 g of Bacto tryptone, 5.0 g of Bacto yeast extract, and 5.0 g of NaCl in 1 litter), but are not limited thereto.
  • a medium containing carbon source, nitrogen source, phosphorus source, and sulfur source, as well as components selected from other various organic components and inorganic components as required can be used. Types and concentrations of the medium components may be arbitrarily determined by those skilled in the art.
  • the carbon source is not particularly limited, so long as the bacterium of the present invention can utilize it to generate heparosan.
  • Specific examples of the carbon source include, for example, saccharides such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and hydrolysates of biomass, organic acids such as acetic acid, fumaric acid, citric acid, succinic acid, and malic acid, alcohols such as glycerol, crude glycerol, and ethanol, and aliphatic acids.
  • a single kind of carbon source may be used, or two or more kinds of carbon sources may be used in combination.
  • the nitrogen source include, for example, ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein decomposition products, ammonia, and urea.
  • ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate
  • organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein decomposition products
  • ammonia and urea.
  • a single kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.
  • the phosphate source include, for example, phosphoric acid salts such as potassium dihydrogenphosphate and dipotassium hydrogenphosphate, and phosphoric acid polymers such as pyrophosphoric acid.
  • phosphoric acid salts such as potassium dihydrogenphosphate and dipotassium hydrogenphosphate
  • phosphoric acid polymers such as pyrophosphoric acid.
  • a single kind of phosphate source may be used, or two or more kinds of phosphate sources may be used in combination.
  • the sulfur source include, for example, inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione.
  • inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites
  • sulfur-containing amino acids such as cysteine, cystine, and glutathione.
  • a single kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.
  • organic components and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing those such as peptone, casamino acid, yeast extract, and soybean protein decomposition product.
  • inorganic salts such as sodium chloride and potassium chloride
  • trace metals such as iron, manganese, magnesium, and calcium
  • vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12
  • amino acids amino acids
  • nucleic acids amino acids
  • nucleic acids amino acids
  • organic components containing those such as peptone, casamino acid, yeast extract, and soybean protein decomposition product.
  • organic components containing those such as peptone, casamino acid, yeast extract, and soybean protein decom
  • auxotrophic mutant that requires an amino acid or the like for growth thereof
  • a gene is introduced by using a vector carrying an antibiotic resistance gene, it is preferable to add the corresponding antibiotic to the medium.
  • Culture conditions are not particularly limited, so long as the bacterium of the present invention can proliferate, and heparosan is produced and accumulated.
  • the culture can be performed with, for example, usual conditions used for culture of bacteria.
  • the culture conditions may be appropriately chosen by those skilled in the art.
  • the culture can be performed, for example, aerobically as aeration culture or shaking culture by using a liquid medium.
  • the culture temperature may be, for example, 30 to 37°C.
  • the culture period may be, for example, 16 to 72 hours.
  • the culture can be performed as batch culture, fed-batch culture, continuous culture, or a combination of these.
  • the culture may be performed as preculture and main culture.
  • the preculture may be performed by using, for example, a plate medium or liquid medium.
  • the method for collecting heparosan from the culture broth is not particularly limited, so long as heparosan can be collected.
  • Examples of the method for collecting heparosan from the culture broth include, for example, the method described in Examples. Specifically, for example, culture supernatant can be separated from the culture broth, and then heparosan contained in the supernatant can be precipitated by ethanol precipitation.
  • the volume of ethanol to be added may be, for example, 2.5 to 3.5 times the volume of the supernatant.
  • the solvent used for precipitating heparosan is not limited to ethanol, and organic solvents miscible with water in an arbitrary ratio can be used.
  • organic solvents examples include methanol, n-propanol, isopropanol, n-butanol, t-butanol, sec-butanol, propylene glycol, acetonitrile, acetone, DMF, DMSO, N-methylpyrrolidone, pyridine, 1,2-dimethoxyethane, 1,4-dioxane, and THF, as well as ethanol.
  • Precipitated heparosan can be dissolved with, for example, water in a volume of 2 times the volume of the original supernatant.
  • the collected heparosan may contain such components as bacterial cells, medium components, moisture, and byproduct metabolites of the bacterium, in addition to heparosan.
  • Heparosan may be purified in a desired degree. Purity of heparosan may be, for example, 30% (w/w) or higher, 50% (w/w) or higher, 70% (w/w) or higher, 80% (w/w) or higher, 90% (w/w) or higher, or 95% (w/w) or higher.
  • heparosan can be detected and quantified by the carbazole method.
  • the carbazole method is a technique widely used as a quantification method for uronic acid, in which a thermal reaction of heparosan and carbazole can be carried out in the presence of sulfuric acid, and absorption at 530 nm of the reaction mixture provided by the generated color substance can be measured to detect and quantify heparosan ( Bitter T. and Muir H.M. (1962) "A modified uronic acid carbazole reaction", Analytical Biochemistry, 4(4):330-334 ).
  • Heparosan can also be detected and quantified by, for example, treating heparosan with heparinase III, which is a heparosan decomposition enzyme, and performing disaccharide composition analysis.
  • Heparin can be produced by using heparosan produced by the bacterium of the present invention. That is, the method for producing heparin of the present invention is a method for producing heparin comprising culturing the bacterium of the present invention in a medium to produce and accumulate heparosan in the medium, chemically and/or enzymatically treating the heparosan to produce heparin, and collecting the heparin. Heparin has an anticoagulant activity, and can be used as an ingredient of drugs.
  • heparin having an anticoagulant activity can be produced ( Zhang Z. et al. (2008) “Solution Structures of Chemoenzymatically Synthesized Heparin and Its Precursors", J. Am. Chem. Soc., 130(39):12998-13007 ).
  • the method for producing heparin may further comprise a depolymerization step.
  • steps as mentioned above for producing heparin from heparosan are also collectively referred to as "heparin production process".
  • the implementation order of the steps in the heparin production process is not particularly limited, so long as heparin having desired properties can be obtained.
  • Heparosan in a state of being contained in the medium may be subjected to the heparin production process, or heparosan collected from the medium may be subjected to the heparin production process. Further, heparosan may be subjected to an arbitrary pretreatment, and then may be subjected to the heparin production process. Examples of the pretreatment include, for example, purification, dilution, concentration, drying, dissolution, and so forth. These pretreatments may also be performed in an appropriate combination. For example, a culture broth containing heparosan as it is, or heparosan purified from such a culture broth to a desired extent may be subjected to the heparin production process.
  • the N-deacetylation can be chemically performed by using, for example, sodium hydroxide.
  • the reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference ( Kuberan B. et al. (2003) "Chemoenzymatic Synthesis of Classical and Non-classical Anticoagulant Heparan Sulfate Polysaccharides", J. Biol. Chem., 278(52):52613-52621 ) can be referred to.
  • the N-sulfation can be chemically performed by using, for example, sulfur trioxide/trimethylamine complex.
  • the reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference ( Kuberan B. et al. (2003) "Chemoenzymatic Synthesis of Classical and Non-classical Anticoagulant Heparan Sulfate Polysaccharides", J. Biol. Chem., 278(52):52613-52621 ) can be referred to.
  • the C5-epimerization can be enzymatically performed by using, for example, C5-epimerase.
  • the C5-epimerase is not particularly limited so long as a C5-epimerase that can catalyze the epimerization of the glucuronic acid (GlcUA) residue into the iduronic acid (IdoA) residue is chosen.
  • a C5-epimerase showing suitable substrate specificity can be chosen and used.
  • the C5-epimerase may be derived from any origin such as animal, plant, and microorganism.
  • C5-epimerase for example, human C5-epimerase can be used.
  • the reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference ( Chen J., et al., "Enzymatic redesigning of biologically active heparan sulfate", J. Biol. Chem., 2005 Dec., 30;280(52):42817-25 ) can be referred to.
  • the 2-O-sulfation can be enzymatically performed by using, for example, a 2-O-sulfation enzyme (2-OST).
  • 2-OST is not particularly limited, so long as 2-OST that can catalyze sulfation of the O-2 position of the IdoA residue is chosen.
  • 2-OST showing suitable substrate specificity can be chosen and used.
  • 2-OST may be derived from any origin such as animal, plant, and microorganism. As 2-OST, for example, hamster 2-OST can be used.
  • the reaction conditions can be appropriately determined by those skilled in the art.
  • the 6-O-sulfation can be enzymatically performed by using, for example, a 6-O-sulfation enzyme (6-OST).
  • 6-OST is not particularly limited so long as 6-OST that can catalyze sulfation of the O-6 position of N-sulfated glucosamine (GlcNS) residue is chosen.
  • GlcNS N-sulfated glucosamine
  • 6-OST showing suitable substrate specificity can be chosen and used.
  • 6-OST may be derived from any origin such as animal, plant, and microorganism. As 6-OST, for example, hamster 6-OST-1 or mouse 6-OST-3 can be used.
  • reaction conditions can be appropriately determined by those skilled in the art. For example, conditions mentioned in the published reference > Chen J., et al., "Enzymatic redesigning of biologically active heparan sulfate", J. Biol. Chem., 2005 Dec., 30;280(52):42817-25 ) can be referred to.
  • the 3-O-sulfation can be enzymatically performed by using, for example, a 3-O-sulfation enzyme (3-OST).
  • 3-OST is not particularly limited so long as 3-OST that can catalyze sulfation of the O-3 position of N-sulfated and 6-O-sulfated glucosamine residue is chosen.
  • 3-OST showing suitable substrate specificity can be chosen and used.
  • 3-OST may be derived from any origin such as animal, plant, and microorganism. As 3-OST, for example, mouse 3-OST-1 can be used.
  • reaction conditions can be appropriately determined by those skilled in the art.
  • conditions mentioned in the published reference Chen J., et al., "Enzymatic redesigning of biologically active heparan sulfate", J. Biol. Chem., 2005 Dec., 30;280(52):42817-25 ) can be referred to.
  • the depolymerization can be performed, for example, by using sulfurous acid or by the photolysis method. Degree of the depolymerization is not particularly limited. The depolymerization may be performed so that heparin having a molecular weight of, for example, 1000 to 35000 Da is produced.
  • the produced heparin can be collected by known methods used for separation and purification of compounds. Examples of such methods include, for example, ion-exchange resin method, membrane treatment, precipitation, and crystallization. These methods can be used in an appropriate combination.
  • the collected heparin may contain such components as component used for the heparin production process, and moisture, in addition to heparin.
  • Heparin may be purified in a desired degree. Purity of heparin may be, for example, 30% (w/w) or higher, 50% (w/w) or higher, 70% (w/w) or higher, 80% (w/w) or higher, 90% (w/w) or higher, or 95% (w/w) or higher.
  • the obtained heparin can be further fractionated to obtain a low molecular weight heparin.
  • Low molecular weight heparin means, for example, a fraction of a molecular weight of 1000 to 10000 Da (average molecular weight, 4000 to 6000 Da).
  • Low molecular weight heparin has an advantage that it shows less adverse reaction of hemorrhage compared with a non-fractionated heparin.
  • Example 1 Construction of heparosan-producing strain from Escherichia coli BL21(DE3) strain
  • the kfiABCD genes ( kfiABCD operon) were cloned into the pVK9 vector (SEQ ID NO: 1, U.S. Published Patent Application No. 20050196846 ) to construct a kfiABCD gene expression plasmid, pVK9-kfiABCD.
  • the PCR cycles consisted of 94°C for 5 minutes, following 30 cycles of 98°C for 5 seconds, 55°C for 10 seconds, and 72°C for 8 minutes, and final maintenance at 4°C. Further, by PCR using pVK9 as the template DNA and the oligonucleotides of SEQ ID NOS: 4 and 5 as the primers, a DNA fragment of pVK9 was obtained.
  • PrimeStar Polymerase (TaKaRa) was used for PCR, and PCR was performed in the reaction composition described in the attached protocol.
  • the PCR cycles consisted of 94°C for 5 minutes, following 30 cycles of 98°C for 5 seconds, 55°C for 10 seconds, and 72°C for 6 minutes, and final maintenance at 4°C.
  • the kfiABCD gene expression plasmid, pVK9-kfiABCD was introduced into the Escherichia coli BL21(DE3) strain (Life Technologies) by electroporation (cell 80 ⁇ L, 200 ⁇ , 25 ⁇ F, 1.8 kV, cuvette 0.1 mL) to obtain Escherichia coli BL21(DE3)/pVK9-kfiABCD strain.
  • This strain was spread on the LB agar medium containing 25 ⁇ g/mL kanamycin, and precultured overnight at 37°C. Then, the cells on the plate were scraped and inoculated into 2 mL of a production medium contained in a test tube. Shaking culture was performed at 37°C for 40 hours, and the culture was finished when glycerol contained in the medium was completely consumed.
  • composition of the production medium is shown below.
  • Component 1 Glycerol 10 g/L
  • Component 2 MOPS (3-N-morpholino-propanesulphonic acid) 41.9 g/L
  • Components 3 Tryptone 8.8 g/L Yeast extract 4.4 g/L Sodium chloride 8.8 g/L
  • the components 1 and 3 were separately sterilized by autoclaving at 120°C for 20 minutes, and the component 2 was sterilized by filter sterilization. After cooling to room temperature, three of the components were mixed.
  • the produced polysaccharides were quantified by the carbazole method ( Bitter, T. and Murir H.M., Anal. Biochem., 1962, 4:330-334 ). The procedures are shown below.
  • the culture supernatant was collected from the culture broth (fermentation broth) by centrifugation.
  • 500 ⁇ L of 100% ethanol was added, and the polysaccharide components were precipitated by centrifugation.
  • the obtained precipitates were air-dried, and dissolved in 300 ⁇ L of 0.2 N aqueous sodium hydroxide solution.
  • the obtained sample (solution, 30 ⁇ L) was calmly added to 150 ⁇ L of sulfuric acid containing 0.025 M tetraboronic acid, and the resulting mixture was heated at 100°C for 10 minutes.
  • Example 2 Structural analysis of produced polysaccharides (2-1) Nuclear magnetic resonance (NMR) spectrum analysis
  • Example 2 The fermentation broth obtained in Example 1 was subjected to bactofugation, and the supernatant was filtered through a 0.45 ⁇ m MF membrane.
  • the obtained filtrate (31 g) was concentrated to 1.1 g by using a UF membrane of 100 KDa (Amicon-15K, 5000 rpm).
  • the concentrate was further washed twice with 40 mL of water.
  • the washed concentrate was further concentrated under reduced pressure in an evaporator, 600 ⁇ L of heavy water was added to the residue to prepare a solution, and then 1 H-NMR measurement was performed.
  • Example 2 The fermentation broth obtained in Example 1 was subjected to bactofugation, and the supernatant was filtered through a 0.45 ⁇ m MF membrane. The obtained filtrate (40 mL) was concentrated to 4 mL by using a UF membrane of 100 KDa (Amicon-15K, 5000 rpm). The concentrate was further washed twice with 40 mL of water.
  • Tri-buffer 200 mM Tri-HCl, 1 M NaCl, 15 mM CaCl 2 , adjusted to pH 7 (25°C) with 35% hydrochloric acid
  • 10 ⁇ L of heparinase III 0.005 unit/mL, produced by Iduron
  • 30 ⁇ L of water were added, and enzyme treatment was performed at 37°C for 16 hours.
  • enzyme-treated mixture 900 ⁇ L of water was added, and used for LC-MS analysis.
  • fragment ions of [m/z] 362 (M+H-H 2 O), 380 (M+H), and 418 (M+K) were detected at a retention time of 6 minutes.
  • the retention time and fragment pattern of the enzyme-treated mixture agreed with the retention time and fragment pattern of a ⁇ GlcUA-GlcNAc standard sample (Heparin disaccharide IV-A sodium salt, Sigma-Aldrich), which is a heparinase decomposition product of heparin and heparan sulfate.
  • the structural formula of the ⁇ GlcUA-GlcNAc standard sample is shown below as the formula (I).
  • Example 2 The fermentation broth obtained in Example 1 was subjected to bactofugation, and the supernatant was filtered through a 0.45 ⁇ m MF membrane.
  • the obtained filtrate (31 g) was concentrated to 1.1 g by using a UF membrane of 100 KDa (Amicon-15K, 5000 rpm).
  • the concentrate was further washed twice with 40 mL of water. GPC measurement of the washed concentrate was performed.
  • retention time peak top
  • Mn number average molecular weight
  • Mw weight average molecular weight
  • Example 3 Screening for factors that improve heparosan-producing ability
  • screening was performed for factors that improve heparosan-producing ability by introducing a genomic library of the Escherichia coli K5 strain into a heparosan-producing strain.
  • Escherichia coli BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain introduced with the kfiABCD gene and showing enhanced expression of the rfaH gene was constructed in accordance with the following procedures.
  • a rfaH gene expression-enhanced strain was obtained by replacing the native promoter region of the rfaH gene on the chromosome with a potent tac promoter ( Amann E. et al. (1983) "Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli", Gene, 25(2-3) :167-78 ) .
  • the rfaH promoter was replaced with the tac promoter by using the method called "Red-driven integration", which was originally developed by Datsenko and Wanner ("One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products", Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645 ). According to this technique, a strain in which a DNA fragment amplified by PCR is inserted into the genomic DNA can be obtained.
  • the primer rfaH-attL Fw shows homology to both a region locating upstream from the rfaH gene, and a region adjacent to the gene that imparts kanamycin (km) resistance existing in the genomic DNA of the NA1 ⁇ c1129 strain.
  • the Km resistance gene kan existing in the genomic DNA of the NA1 ⁇ c1129 strain is inserted between the attL and attR genes, which are the attachment sites of ⁇ phage, and the tac promoter (Ptac, SEQ ID NO: 8) is inserted further downstream therefrom in the order of attL-kan-attR-Ptac.
  • the primer rfaH-Ptac Rv shows homology to both the rfaH region and a region locating downstream from the tac promoter in the genomic DNA of the NA1 ⁇ c1129 strain.
  • the plasmid pKD46 contains the genes of the ⁇ Red homologous recombination system ( ⁇ , ⁇ , and exo genes), i.e.
  • phage ⁇ a 2,154 nucleotide DNA fragment of phage ⁇ (GenBank/EMBL Accession No. J02459, nucleotide positions 31088 to 33241), under the control of the arabinose-inducible P araB promoter.
  • the plasmid pKD46 is necessary for integration of the PCR product into the chromosome of the BL21(DE3) strain.
  • the Escherichia coli BL21(DE3)/pKD46 strain was grown overnight at 30°C in the LB medium containing ampicillin (100 mg/L). This culture was diluted 100 times with the LB medium (100 mL) containing ampicillin and L-arabinose (1 mM).
  • the cells were grown at 30°C with aeration until OD600 became about 0.3, then concentrated 100 times, washed 3 times with ice-cooled aqueous glycerol solution (10%), and thereby made into electrocompetent cells. Electroporation was performed by using 70 ⁇ l of the competent cells and about 100 ng of the PCR product. After the electroporation, the cells were incubated in 1 mL of the SOC medium ( Molecular Cloning A Laboratory Manual, 2nd edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989 )) at 37°C for 2.5 hours, applied to the LB agar medium, and grown at 37°C to select Km resistant strains.
  • SOC medium Molecular Cloning A Laboratory Manual, 2nd edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989 )
  • the substitution of the tac promoter for the rfaH promoter was confirmed by PCR using the primer rfaH CF (SEQ ID NO: 9) and primer rfaH CR (SEQ ID NO: 10), which are specific to the nucleotide sequence after the promoter substitution.
  • PrimeStar Polymerase was used for PCR.
  • the PCR cycles consisted of 94°C for 5 minutes, following 30 cycles of 98°C for 5 seconds, 55°C for 10 seconds, and 72°C for 2 minutes, and final maintenance at 4°C.
  • a strain in which amplification of a DNA fragment of 1.6 kbp could be confirmed was designated as BL21(DE3)-Ptac-rfaH(KmR) strain.
  • plasmid pMW118-int-xis (ampicillin resistant (AmpR)) was introduced ( WO2005/010175 ) to the strain.
  • AmpR clones were grown at 30°C on an LB agar plate containing 150 mg/L of ampicillin. Several tens of AmpR clones were picked up, and a Km-sensitive strain was selected. By incubating the Km sensitive strain at 42°C on an LB agar plate, the plasmid pMW118-int-xis was removed from the Km-sensitive strain.
  • An obtained Amp sensitive strain was designated as BL21(DE3)-Ptac-rfaH strain.
  • the plasmid pVK9-kfiABCD produced in Example 1 was introduced into the BL21(DE3)-Ptac-rfaH strain by electroporation to obtain BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain.
  • Culture was performed in test tubes by using the same medium and culture method as those of Example 1, and heparosan production amount was determined by the carbazole method.
  • the heparosan production amounts of the BL21(DE3)/pVK9-kfiABCD strain of which expression of the rfaH gene was not enhanced, and the BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain of which expression of the rfaH gene was enhanced are shown in Table 4.
  • Table 4 Heparosan production amount of BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD 290.4 ⁇ 32.7 BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD 506.2 ⁇ 69.9
  • Fragments of the genomic DNA of the Escherichia coli K5 strain were cloned into the pSTV28 vector (SEQ ID NO: 11, TaKaRa) to constructed genomic library.
  • the details of the construction of the genomic library are shown below.
  • the genomic DNA of the Escherichia coli K5 strain (3 ⁇ g) was randomly fragmented by using a DNA fragmentation apparatus (Hydro-Shear, Gene Machine), and fractionated by agarose electrophoresis. A portion containing DNAs of about 3 to 5 kb was cut out from the agarose gel, and DNAs were extracted, purified, and then blunt-ended. Then, the genomic DNA fragments were ligated with 50 ng of the plasmid vector pSTV28 (TaKaRa) digested with Hinc II and dephosphorylated with Alkaline Phosphatase ( E. coli C75) (TaKaRa).
  • the Escherichia coli HST08 strain (TaKaRa) was transformed with the ligation product by electroporation. Seventy percent or more of the obtained transformants contained inserts of about 3 to 5 kb. The transformants were cultured, and the plasmids were extracted to obtain a genomic library.
  • the genomic library or pSTV28 as a control was introduced into the BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain by electroporation.
  • One clone was selected from each of the obtained genomic library transformants, and used to perform fermentative production culture. Media of the following compositions were used for the culture.
  • the seed medium was sterilized by autoclaving at 120°C for 20 minutes.
  • Component 1 Glycerol 10 g/L
  • Component 2 MOPS (3-N-morpholino-propanesulphonic acid) 41.9 g/L
  • Components 3 Tryptone 8.8 g/L Yeast extract 4.4 g/L Sodium chloride 8.8 g/L
  • the components 1 and 3 were separately sterilized by autoclaving at 120°C for 20 minutes, and the component 2 was sterilized by filter sterilization. After cooling to room temperature, three of the components were mixed.
  • the heparosan production culture was performed according to the following procedures. First, one colony of each transformant was inoculated into each well of a 96-well plate (MEDISCAN), which contained 750 ⁇ L of the seed medium, and shaking culture was performed overnight at 37°C on a shaking machine (Tietech). Then, 20 ⁇ L of the seed culture broth was inoculated into 2 mL of the production medium contained in a test tube, shaking culture was performed at 37°C for 30 hours, and the culture was terminated when the glycerol in the medium was completely consumed. In order to make the cells harbor the plasmids, kanamycin (25 mg/L) and chloramphenicol (25 mg/L) were added to the medium over the whole culture period.
  • Heparosan produced in the medium was quantified by the carbazole method ( Bitter, T. and Murir H.M., Anal. Biochem., 1962, 4:330-334 ). There were isolated clones that showed increased heparosan accumulation amounts as compared with the simultaneously cultured control vector (pSTV28)-introduced strain.
  • the nucleotide sequences of the inserted DNA fragments were determined by using the primer pSTV Fw (SEQ ID NO: 12) and primer pSTV Rv (SEQ ID NO: 13).
  • the respective plasmids contained rbsBKR-hsrA, glgBX, ybiXIJCB, rcsBD-micF, pcoESR, yhcNO-aaeBAX, g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB, thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, irp (part), bhsA-ycfS, lepB-rnc-era, dapA-gcvR-bcp-hyfA, rpoE-nadB-yfiC-srmB, g1414-g1413, nu
  • the irp means a part of the irp2 gene and a part of the irp1 gene.
  • the rfaH gene was cloned from the Escherichia coli BL21(DE3) strain into pMIV-Pnlp0-ter to construct a rfaH gene expression plasmid, pMIV-Pnlp0-rfaH.
  • pMIV-Pnlp0-ter contains the potent nlp0 promoter (Pnlp0) and the rrnB terminator, and the promoter and the terminator can function as an expression unit of a target gene when the target gene is inserted therebetween.
  • Pnlp0 means the wild-type promoter of the nlpD gene of the Escherichia coli K-12 strain.
  • the PCR cycles consisted of 95°C for 3 minutes, following 2 cycles of 95°C for 60 seconds, 50°C for 30 seconds, and 72°C for 40 seconds, 25 cycles of 94°C for 20 seconds, 55°C for 20 seconds, and 72°C for 15 seconds, and 72°C for 5 minutes as the final cycle.
  • the obtained fragment was treated with Sal I and Pae I, and inserted into pMIV-5JS (Japanese Patent Laid-open (Kokai) No. 2008-99668 ) at the Sal I- Pae I site to obtain plasmid pMIV-Pnlp0.
  • the nucleotide sequence of the Pae I- Sal I fragment of the Pnlp0 promoter inserted into this pMIV-Pnlp0 plasmid is as shown as SEQ ID NO: 16.
  • the PCR cycles consisted of 95°C for 3 minutes, following 2 cycles of 95°C for 60 seconds, 50°C for 30 seconds, and 72°C for 40 seconds, 25 cycles of 94°C for 20 seconds, 59°C for 20 seconds, and 72°C for 15 seconds, and 72°C for 5 minutes as the final cycle.
  • the obtained fragment was treated with Xba I and Bam HI, and inserted into pMIV-Pnlp0 at the Xba I- Bam HI site to obtain plasmid pMIV-Pnlp0-ter.
  • a rfaH gene fragment was obtained.
  • the sites for the restriction enzymes Sal I and Xba I were designed in the 5' end regions of the respective primers.
  • PrimeStar Polymerase was used for PCR, and the PCR cycles consisted of 94°C for 5 minutes, following 30 cycles of 98°C for 5 seconds, 55°C for 10 seconds, and 72°C for 4 minutes, and final maintenance at 4°C.
  • the obtained fragment was treated with Sal I and Xba I, and inserted into pMIV-Pnlp0-ter at the Sal I -Xba I site to obtain plasmid pMIV-Pnlp0-rfaH.
  • an rfaH expression unit comprising the nlpD promoter, rfaH gene, and rrnB terminator connected in this order in the pMIV-5JS vector.
  • the nucleotide sequence of the rfaH gene of the Escherichia coli BL21(DE3) strain cloned in this experiment is shown as SEQ ID NO: 46.
  • Table 7 Effect of enhancement of rfaH gene expression in BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pMIV-5JS 376.9 ⁇ 48.3 BL21(DE3)/pVK9-kfiABCD/pMIV-Pnlp0-rfaH 857.9 ⁇ 219.1
  • Example 11 Heparosan production using rpoE gene expression-enhanced strain
  • the rpoE gene was cloned from the Escherichia coli K5 strain into pMIV-Pnlp8-ter to construct a rpoE gene expression plasmid, pMIV-Pnlp0-rpoE.
  • pMIV-Pnlp0-ter contains the potent nlp8 promoter (Pnlp8), and the promoter and a terminator can function as an expression unit of a target gene when the target gene is inserted therebetween.
  • Pnlp8 means a variant promoter of the nlpD gene of the Escherichia coli K-12 strain.
  • the details of the construction of the expression vector pMIV-Pnlp8-ter are shown below.
  • the wild-type nlpD promoter PnlpO
  • the wild-type nlpD promoter region ( Fig. 1 , SEQ ID NO: 165) contains two regions presumed to function as a promoter, and they are indicated as Pnlp1 and Pnlp2, respectively, in the drawing.
  • the obtained fragments for the 3' end side and 5' end side were ligated by using the Bgl II sites designed in the primers P7 and P8 to construct a DNA fragment containing a variant nlpD promoter in full length, of which two -10 regions were randomized.
  • PCR using this DNA fragment as the template, as well as the primer P1 and primer P2, the DNA fragment containing the full length of the variant nlpD promoter was amplified.
  • the PCR cycles consisted of 95°C for 3 minutes, following 2 cycles of 95°C for 60 seconds, 50°C for 30 seconds, and 72°C for 40 seconds, 12 cycles of 94°C for 20 seconds, 60°C for 20 seconds, and 72°C for 15 seconds, and 72°C for 5 minutes as the final cycle.
  • the amplified DNA fragment containing the full length of the variant nlpD promoter was treated with the restriction enzymes SalI and PaeI designed in the 5' end regions of the primers, and inserted into the plasmid pMIV-Pnlp0-ter similarly treated with Sal I and Pae I to replace the wild-type nlpD promoter (Pnlp0) on the plasmid with the variant nlpD promoter.
  • plasmids obtained as described above one having the promoter sequence shown in Fig. 2 (Pnlp8, SEQ ID NO: 168) was chosen, and designated as pMIV-Pnlp8-ter.
  • the nucleotide sequence of the Pae I- Sal I fragment of the Pnlp8 promoter inserted into this plasmid was as shown as SEQ ID NO: 169.
  • rpoE gene expression plasmid pMIV-Pnlp8-rpoE
  • pMIV-Pnlp8-rpoE The details of the construction of the rpoE gene expression plasmid, pMIV-Pnlp8-rpoE, are described below.
  • PCR using the chromosomal DNA of the Escherichia coli K5 strain as the template, as well as the primer rpoE-SalI Fw (SEQ ID NO: 170) and primer rpoE-xba Rv (SEQ ID NO: 171), a DNA fragment of the rpoE gene was obtained.
  • PrimeStar Polymerase (TaKaRa) was used for PCR, and PCR was performed in the reaction composition described in the attached protocol.
  • PCR cycles consisted of 94°C for 5 minutes, following 30 cycles of 98°C for 5 seconds, 55°C for 10 seconds, and 72°C for 2 minutes, and final maintenance at 4°C. Further, by PCR using pMIV-Pnlp8-ter as the template DNA, as well as the oligonucleotides of SEQ ID NOS: 172 and 173 as the primers, a DNA fragment of pMIV-Pnlp8-ter was obtained.
  • PrimeStar Polymerase (TaKaRa) was used for PCR, and PCR was performed in the reaction composition described in the attached protocol.
  • the PCR cycles consisted of 94°C for 5 minutes, following 30 cycles of 98°C for 5 seconds, 55°C for 10 seconds, and 72°C for 6 minutes, and final maintenance at 4°C. Both the obtained DNA fragments were ligated by using In-Fusion (registered trademark) HD Cloning Kit (Clontech) to construct an rpoE gene expression plasmid, pMIV-Pnlp8-rpoE.
  • the nucleotide sequence of the cloned rpoE gene is shown as SEQ ID NO: 174.
  • Table 13 Effect of enhancement of rpoE gene expression in BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pMIV-5JS 96.1 ⁇ 5.8 BL21(DE3)/pVK9-kfiABCD/pMIV-Pnlp8-rpoE 183.6 ⁇ 7.8
  • heparosan-producing ability of bacteria can be improved, and heparosan can be efficiently produced.

Claims (6)

  1. Escherichia-Bakterium mit einer Heparosan-produzierenden Fähigkeit, worin das Bakterium so modifiziert wurde, dass die Expression des rpoE-Gens im Vergleich zu einem nicht modifizierten Stamm erhöht wird.
  2. Bakterium nach Anspruch 1, worin die Expression des rpoE-Gens durch Erhöhen der Kopienzahl des rpoE-Gens und/oder Modifizieren einer Genexpressionskontrollsequenz des rpoE-Gens erhöht wird.
  3. Bakterium nach Anspruch 1 oder 2, das Escherichia coli ist.
  4. Bakterium nach einem der Ansprüche 1 bis 3, worin das rpoE-Gen eine DNA, die die komplementäre Sequenz der Nukleotidsequenz der Positionen 355 bis 930 der SEQ ID Nr. 116 umfasst, oder eine DNA ist, die eine Nukleotidsequenz umfasst, die eine Identität von 90% oder höher zur komplementären Sequenz der Nukleotidsequenz der Positionen 355 bis 930 der SEQ ID Nr. 116 zeigt und eine Eigenschaft aufweist, die Heparosan-produzierende Fähigkeit eines Escherichia-Bakteriums mit Heparosan-produzierender Fähigkeit zu erhöhen, wenn die Expressionsmenge davon in dem Bakterium erhöht wird.
  5. Verfahren zur Herstellung von Heparosan, wobei das Verfahren umfasst:
    Kultivieren des Bakteriums nach einem der Ansprüche 1 bis 4 in einem Medium, um Heparosan in dem Medium herzustellen und anzureichern; und
    Sammeln des Heparosans aus dem Medium.
  6. Verfahren zur Herstellung von Heparin, wobei das Verfahren umfasst:
    Kultivieren des Bakteriums nach einem der Ansprüche 1 bis 4 in einem Medium, um Heparosan in dem Medium zu produzieren und anzureichern;
    chemisches und/oder enzymatisches Behandeln des Heparosans zur Herstellung von Heparin; und
    und Sammeln des Heparins.
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US20160201103A1 (en) 2016-07-14
EP3054005A4 (de) 2017-09-13
JPWO2015050184A1 (ja) 2017-03-09
US20180237479A1 (en) 2018-08-23
JP6569530B2 (ja) 2019-09-04
WO2015050184A1 (ja) 2015-04-09
EP3620525A1 (de) 2020-03-11
US9975928B2 (en) 2018-05-22
EP3054005A1 (de) 2016-08-10
US10611804B2 (en) 2020-04-07

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